This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. CHESS has been an essential resource in the Arnold laboratory's effort to study the structure and function of reverse transcriptase (RT), a key component of the AIDS virus and the target of many of the most widely used anti-AIDS drugs. The Arnold group has solved three-dimensional structures of wild-type and drug-resistant HIV-1 RT in complexes with a variety of antiviral drugs and model segments of the HIV genome. These studies, together with contributions from other laboratories, have yielded numerous insights into polymerase structure-function relationships, detailed mechanisms of drug resistance, and provided the basis for structure-based design of RT inhibitors. A collaboration between the Arnold laboratory and the Janssen/Tibotec group led to the development of a number of inhibitors that show great promise as potential treatments for AIDS, two of which are currently in Phase II (TMC278) and Phase III (TMC125) clinical trials in the United States and overseas [165-167]. Structural studies of HIV-1 RT complexed with RNase H inhibitors [168] are also being pursued;the RNase H activity of RT is also essential for HIV replication, yet no drugs targeting RNase H have been developed. During the past year remarkable success has been achieved in terms of obtaining high-resolution diffraction from HIV-1 RT specifically engineered to yield novel crystal forms. Diffraction data extending to 1.8 [unreadable] resolution have been obtained for engineered HIV-1 RT in complex with the Janssen inhibitor TMC278. The engineering strategy has consisted of making systematic variations of the N- and Ctermini of the HIV-1 RT heterodimer in a coexpressed p66/p51 system, with a removable His-tag for convenient purification, and selected surface mutations such as Lys=>Ala. Among the noteworthy aspects of the high resolution HIV-1 RT/TMC278 structure (J. Bauman, K. Das et al., in preparation) is that this critical structure had been elusive despite literally thousands of crystallization attempts with this complex. An important implication of this new HIV-1 RT construct and crystal form is that further structurebased drug design for HIV-1 RT inhibitors can proceed both more accurately and rapidly. They have been able to collect numerous datasets extending to 2 [unreadable] resolution or better with and without bound inhibitors and are now searching for higher resolution crystal forms that can accommodate nucleic acid binding. Many crystal forms were evaluated using X-rays from the CHESS F1 and A1 beamlines in the process of identifying several engineered RT constructs that yielded high-resolution crystals. Prior to this work, the highest published resolution of any HIV-1 RT structure was 2.2 [unreadable], and most published RT structures are in the 2.7-3.0 [unreadable] range. The development of high-through put capability at CHESS, and in particular, the use of the new robotic automounter at CHESS F1, has made it possible to evaluate a large number of candidate crystals and will continue to be an extremely valuable resource as the Arnold group carries out a systematic screen for the binding of drug-like fragments to RT. These studies are expected to lead to the identification of potential new target sites as well as new leads for inhibitor development.